A major focus of HIV-1 vaccine efforts is the elicitation of antibodies capable of neutralizing diverse circulating strains of the virus. The isolation of broadly neutralizing antibodies from HIV-infected adults and identification of conserved sites of vulnerability on the viral envelope targeted by these antibodies has added momentum to the search for an antibody-based vaccine. However, detailed studies of broad HIV-specific monoclonal antibodies from adults have also revealed potential obstacles to the elicitation of broad responses by immunization, such as the requirement for long-term antibody maturation. It was recently shown that infants develop plasma neutralization breadth more rapidly than adults, and infant responses do not appear to be mediated by a dominant broadly neutralizing antibody of known specificity. The characteristics of infant HIV- specific antibodies contributing to plasma neutralization breadth have not yet been studied, but may provide unique insights into the process of developing HIV-specific broadly neutralizing antibody responses. The proposed project utilizes samples from a unique cohort of HIV-positive infants who were part of a study conducted from 1992 to 1998 in Nairobi, Kenya and identified as developing plasma neutralization breadth. Infant HIV-specific neutralizing antibodies will be isolated and characterized to address the hypothesis that infant plasma neutralization breadth is mediated by polyclonal antibodies targeting known epitopes and exhibiting lower levels of maturation compared to adult broadly neutralizing antibodies. The first part of this proposal combines exciting new methods for cloning antibodies from HIV-specific single B cells: an infant- derived envelope trimer as bait and large-scale culture of memory B cells and functional screening by high- throughput neutralization assays. Defining the ability of isolated antibodies to neutralize diverse viral variants in the second part of this proposal will establish whether a monoclonal antibody or a combination of polyclonal antibodies account for infant plasma neutralizing activity. Lastly, the third part of this proposal will define sites on the viral envelope targeted y infant antibodies by identifying HIV-1 envelope mutations conferring resistance to isolated antibodies. Better understanding infant broadly neutralizing antibody responses may inform vaccine strategies to elicit them without the requirement for long-term antibody maturation. Furthermore, identifying the targets of infant-derived antibodies will inform vaccine immunogen design.